敲除LTA4H基因的PK-15细胞系构建及其对口蹄疫病毒复制的影响  

Construction of PK-15 cell line with LTA4H gene knockout and its impact on the replication of foot-and-mouth disease virus

作　　者：王家丽 蒲秀瑛[1] 杨帆[2,3] 邵文华 黄梦瑶 曹伟军 郑海学[1,2,3] 张伟 WANG Jiali;PU Xiuying;YANG Fan;SHAO Wenhua;HUANG Mengyao;CAO Weijun;ZHENG Haixue;ZHANG Wei(School of Life Science and Engineering,Lanzhou University of Technology,Lanzhou 730050,Gansu,China;State Key Laboratory of Animal Disease Prevention and Control,Lanzhou Veterinary Institute,Chinese Academy of Agricultural Sciences/College of Animal Medicine and Biosafety,Lanzhou University,Lanzhou 730000,Gansu,China;Gansu Provincial Research Center of Pathogen Biology,Lanzhou 730046,Gansu,China)

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730000 [3]甘肃省病原生物学基础学科研究中心,甘肃兰州730046

出　　处：《微生物学报》2024年第3期733-744,共12页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32102639,32072831);国家重点研发计划(2021YFD1800300);甘肃省杰出青年基金(21JR7RA026);兰州大学中央高校基本科研业务费专项资金(lzujbky-2022-ey20);“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2023SDZG02);国家生猪产业技术体系(CARS-35)。

摘　　要：【目的】利用CRISPR/Cas9基因编辑技术构建白三烯A4水解酶(leukotriene A4 hydrolase,LTA4H)基因缺失的猪肾细胞系(porcine kidney-15,PK-15),探究LTA4H对口蹄疫病毒(foot-and-mouth disease virus,FMDV)复制的影响,为开展LTA4H功能研究及调控病毒复制机制研究提供理论依据。【方法】设计2条针对猪LTA4H基因的引导RNA(small guide RNA,sgRNA),分别构建至载体pX459-puro-MCS中;将CRISPR重组质粒转染PK-15细胞,用嘌呤霉素(puromycin)抗生素筛选,并通过有限稀释法筛选单克隆细胞,之后通过Western blotting和测序检测LTA4H基因的敲除,获得LTA4H基因功能缺失细胞系。使用Western blotting、RT-qPCR及病毒滴度测定等方法检测敲除LTA4H基因后对FMDV复制及相关蛋白的表达情况。【结果】获得的单克隆敲除细胞系与野生型细胞相比,能够显著抑制FMDV复制。【结论】本研究成功构建了LTA4H基因敲除的PK-15细胞系,证明LTA4H对FMDV的复制具有促进作用,研究结果为后续LTA4H功能研究提供理论依据。[Objective]A LTA4H-deleted porcine kidney cell line(PK-15)was constructed by CRISPR/Cas9 to study the effect of LTA4H on the replication of foot-and-mouth disease virus(FMDV),with a view to providing a theoretical basis for revealing the functions of LTA4H and the mechanism of regulating virus replication.[Methods]Two small guide RNAs(sgRNAs)targeting porcine LTA4H were designed and integrated into the vector pX459-puro-MCS,respectively.The recombinant plasmid was transfected into PK-15 cells,which were then cultured in the medium supplemented with puromycin,and the monoclonal cells were selected by limiting dilution method.Western blotting and sequencing were performed to detect LTA4H knockout,and LTA4H-deleted cells were obtained.Furthermore,Western blotting,RT-qPCR,and virus titer assay were employed to examine FMDV replication and protein expression after LTA4H knockout.[Results]Compared with the wild type cells,the LTA4H-deleted PK-15 cells exhibited inhibited FMDV replication.[Conclusion]This study successfully constructed a PK-15 cell line with LTA4H gene knockout,demonstrating that LTA4H can promote the replication of FMDV,and the results provided a theoretical basis for the subsequent research on the function of LTA4H.

关 键 词：CRISPR/Cas9 白三烯A4水解酶 细胞系 口蹄疫病毒 

分 类 号：S852.65[农业科学—基础兽医学]